Chiral diacylhydrazine ligands for modulating the expression of exogenous genes via an ecdysone receptor complex

1. A method of modulating the expression of a gene of interest in a host cell, wherein the host cell comprises a first recombinant gene expression cassette comprising a first polynucleotide encoding a first polypeptide comprising:

i) a DNA-binding domain; and

ii) a Group H nuclear receptor ligand binding domain;

and a second recombinant gene expression cassette comprising:

i) a response element capable of binding to said DNA binding domain;

ii) a promoter; and

iii) said gene of interest;

the method comprising contacting said host cell with a compound selected from the group consisting of:

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide; and

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide,

wherein the expression of the gene of interest is modulated.

2. A method of modulating the expression of a gene in a host cell comprising the steps of:

(a) introducing into the host cell a gene expression modulation system comprising:

(i) a first recombinant gene expression cassette that is capable of being expressed in a host cell, said first recombinant gene expression cassette comprising a polynucleotide sequence that encodes a first hybrid polypeptide comprising:

(a) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and

(b) an ecdysone receptor ligand binding domain;

(ii) a second recombinant gene expression cassette that is capable of being expressed in the host cell, said second recombinant gene expression cassette comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising:

(a) a transactivation domain; and

(b) a chimeric retinoid X receptor ligand binding domain; and

(iii) a third recombinant gene expression cassette that is capable of being expressed in a host cell, said third recombinant gene expression cassette comprising a polynucleotide sequence comprising:

(a) a response element recognized by the DNA-binding domain of the first hybrid polypeptide;

(b) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and

(c) a gene whose expression is to be modulated; and

(b) contacting the host cell with a compound selected from the group consisting of:

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide; and

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide,

wherein the expression of the gene in the host cell is modulated.

3. A method for producing a polypeptide comprising the steps of:

(a) selecting a host cell which is substantially insensitive to exposure to a compound selected from the group consisting of:

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N′-benzoyl-N-(1-tert-butyl-butyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N′-(2-bromo-benzoyl)-N-(1-tert-butyl-butyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methyl-benzoyl)-hydrazide;

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide; or

(R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide,

(b) introducing into the host cell:

(1) a DNA construct comprising:

(a) an exogenous gene encoding the polypeptide; and

(b) a response element;

wherein the exogenous gene is under the control of the response element; and

(ii) an ecdysone receptor complex comprising:

(a) a DNA binding domain that binds to the response element;

(b) a binding domain for said compound; and

(c) a transactivation domain; and

(c) exposing the cell to said compound; wherein a polypeptide is produced.

4. The method of claim 1 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide.

5. The method of claim 1 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide.

6. The method of claim 1 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide.

7. The method of claim 1 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide.

8. The method of claim 1 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.

9. The method of claim 2 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide.

10. The method of claim 2 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N-(2-methyl-benzoyl)-hydrazide.

11. The method of claim 2 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide.

12. The method of claim 2 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide.

13. The method of claim 2 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.

14. The method of claim 3 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N-(2-ethyl-3-methoxy-benzoyl)-hydrazide.

15. The method of claim 3 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-methyl-benzoyl)-hydrazide.

16. The method of claim 3 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-fluoro-benzoyl)-hydrazide.

17. The method of claim 3 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tort-butyl-butyl)-N′-(4-methyl-benzoyl)-hydrazide.

18. The method of claim 3 , comprising contacting said host cell with (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide.

19. The method of claim 1 , wherein said gene of interest is IL-12.

20. The method of claim 2 , wherein said gene whose expression is to be modulated is IL-12.

21. The method of claim 3 , wherein said polypeptide is IL-12.